The thyroid cell monolayer in culture

When cultured on collagen coated nitrocellulose filters, thyroid epithelial cells form morphologically and functionally polarized monolayers. The bioelectric parameters of these monolayers were measured after mounting in Ussing chambers; transepithelial potential (V _ab), short circuit current (I _sc) and transepithelial resistance were respectively 12±1 mV (apical side negative), 3.8±0.2 A cm^–2 and 3250±214 cm² (mean±SEM,n=75). Eighty two percent of the short circuit current was related to sodium absorption as shown by inhibition by apical amiloride (K _m=0.2 M) and by basal ouabain (K _1/2=0.3 M). Amphotericin B (5–25 g/ml) added to the apical bath increasedI _sc suggesting an apical rate-limiting step. Step by step replacement of choline by Na⁺ in a Na⁺-free medium resulted in a progressive increase inV _ab andI _sc with half maximal effect at 20±1 mM Na⁺. Thyrotropin (TSH) increasedI _sc andV _ab in a biphasic way with a transient maximum after 5 min and a plateau after 20 min (about four times the basal level at 100 U/ml TSH). This increase in sodium transport was also inhibited by apical amiloride. Thus, in culture, the thyroid cell monolayer behaves as a tight sodium absorbing epithelium controlled by TSH, with a rate limiting apical sodium channel as the entry mechanism and a basolateral Na⁺, K⁺-ATPase as the electromotive force.     

Transepithelial dipeptide (glycylsarcosine) transport across epithelial monolayers of human Caco-2 cells is rheogenic

Net transepithelial transport (and cellular accumulation) of the dipeptide glycylsarcosine (Gly-Sar), across the apical membrane of human intestinal Caco-2 epithelia, is driven by a proton gradient (Na⁺-free conditions) and displays saturation kinetics (K_m 17.4±5.1 mM, V_max of 92.8±15.6 nmol.cm^–2.h^–1). Net Gly-Sar transport is associated with the stimulation of an inward short-circuit current (I_sc). This dipeptide-stimulated I_sc is observed in both Na⁺-containing and Na⁺-free conditions, is stimulated by apical acidity, and displays saturation kinetics (in Na⁺-free media at apical pH 6.0, K_m of 13.6±4.5 mM and a V_max of 284.1±39.3 nmol.cm^–2.h^–1). The maximal capacities of Gly-Sar transport and I_sc suggest a dipeptide/proton stoichiometry greater than unity (13).     

Microelectrode study of voltage-dependent Ba2+ and Cs+ block of apical K+ channels in the skin of Rana temporaria

The blockage of the apical K⁺ channels in frog species Rana temporaria by Ba²⁺ and Cs⁺ is strongly voltage-dependent. The interaction of both blockers with the K⁺ channels was studied by recording relations between the K⁺ currents (I _K) and the transepithelial and intracellular potential. Mucosal Ba²⁺ and Cs⁺ depress I _K, hyperpolarize the cell and induce pronounced nonlinearities in the current/voltage (I/V) relations. The nonlinearities are caused by the voltage-dependent interaction of Ba²⁺ and Cs⁺ with the binding site. Consequently, the apical membrane resistance not only depends on the blocker concentration but also on the apical membrane potential. Also the fractional resistance, fR _a, and the voltage divider ratio, fV _a, will change with blocker concentration and voltage. Owing to this non-ohmic behaviour, measurements of fV _a in the presence of Ba²⁺ deviate markedly from the expected fR _a values. The inhibitory effect of Ba²⁺ and Cs⁺ was analysed at different transepithelial and apical membrane voltages. The relation between the Michaelis-Menten constants and the voltage could be fitted with equations based on Eyring rate theory with the assumption of a single binding site. With this model we calculated the relative electrical position of the binding site for the blocker (), referred to the extracellular side of the channel. We obtained for Ba²⁺, =0.34±0.05 and for Cs⁺, =0.81±0.01. Comparison of the results from apical and transepithelial I/V relations demonstrates that the analysis of the transepithelial data provides overestimated values of the Hill coefficient and results in an underestimation of .     

Current-voltage relations of Cs+-inhibited K+ currents through the apical membrane of frog skin

The voltage-dependence of the inhibitory effect of mucosal Cs⁺ on the inward K⁺ current through the apical membrane of frog skin (Rana temporaria) was studied by recording transepithelial current-voltage relations. Experiments were performed with skins exposed to NaCl and KCl Ringer solutions on the serosal and mucosal side respectively (contron skins), as well as with tissues incubated with K₂SO₄ Ringer solutions on both sides (depolarized skins). Studies of the dose-depedence of the Cs⁺ block showed that under both experimental conditions the apparent affinity of Cs⁺ increased as the transepithelial potential was clamped at higher mucosal positive voltages. Under control conditions, the concentration of Cs⁺ required to block 50% of the K⁺ current (K_Cs) recorded while the transepithelial voltage was clamped at zero mV was 16 mmol/1. K_Cs decreased exponentially with muscosal positive voltages. The dependence of K_Cs on the membrane potential was analyzed with Eyring rate theory in which Cs⁺ was assumed to block the K⁺ transport by binding to a site within the channel. The analysis showed that this site is located at a relative electrical distance =0.32 of the voltage drop across the apical membrane, measured from the cytosolic side. The Hill coefficient obtained from this analysis wasn=3.1. Experiments with K⁺-depolarized tissues showed that only inward K⁺ currents recorded with positive transepithelial voltages were depressed by external Cs⁺. Also under these conditions K_Cs showed an exponential dependence on the transepithelial potential. The analysis of these data with the rate theory revealed =0.09 andn=1.7. The difference in found in control and depolarized tissues can be explained by the influence of the basolateral membrane resistance on theI–V relations.     

Effects of dopamine on ion transport across the rat distal colon

Dopamine (5·10^–6–5·10^–4 M) induced a concentration-dependent decrease in short-circuit current (I_sc) across the rat distal colon. This response was preceded by a transient and inconsistent increase in I_sc. The -adrenoceptor blocker phentolamine and the inhibitors of dopamine-2-like (D₂-like) receptors L-741,626 and L-745,870 inhibited the dopamine response, suggesting a contribution of adrenergic and dopaminergic receptors. The decrease in I_sc evoked by dopamine was inhibited by bumetanide, an inhibitor of the basolateral Na⁺-K⁺-2 Cl^– cotransporter responsible for the uptake of K⁺, and by quinine, a blocker of apical K⁺ channels, indicating that stimulation of K⁺ secretion contributes to the measured change in I_sc. In patch-clamp experiments dopamine hyperpolarized the membrane and increased cellular K⁺ current. This response was not concomitant with a change in the intracellular [Ca²⁺] as demonstrated in parallel fura-2 experiments. These results demonstrate that dopamine, like other catecholamines, stimulates colonic K⁺ secretion.     

K+ recirculation in A6 cells at increased Na+ transport rates

Homocellular regulation of K⁺ at increased transcellular Na⁺ transport implies an increase in K⁺ exit to match the intracellular K⁺ load. Increased K⁺ conductance, g_K, was suggested to account for this gain. We tested whether such a mechanism is operational in A6 monolayers. Na⁺ transport was increased from 5.1±1.0 A/cm² to 20.7±1.3 A/cm² by preincubation with 0.1 mol/l dexamethasone for 24 h. Basolateral K⁺ conductances were derived from transference numbers of K⁺, t _K, and basolateral membrane conductances, g_b, using conventional microelectrodes and circuit analysis with application of amiloride. Activation of Na⁺ transport induced an increase in g_b from 0.333±0.067 mS/ cm² to 1.160±0.196 mS/cm² and t _K was reduced to 0.22±0.01 from a value of 0.70±0.05 in untreated control tissues. As a result, g_K remained virtually unchanged at increased Na⁺ transport rates. The increase in g_b after dexamethasone was due to activation of a conductive leak pathway presumably for Cl^–. Increased K⁺ efflux, I _K, was a consequence of the larger driving force for K⁺ exit due to depolarization at an elevated Na⁺ transport rate. The relationship between calculated K⁺ fluxes and Na⁺ transport rate, measured as the I _sc, is described by the linear function I _K=0.624×I _Na–0.079, which conforms with a stoichiometry 23 for the fluxes of K⁺ and Na⁺ in the Na⁺/K⁺-ATPase pathway. Our data show that homocellular regulation of K⁺ in A6 cells is not due to up-regulation of g _K .     

Regulatory volume decrease in a renal distal tubular cell line (A6) II. Effect of Na+ transport rate

A6 epithelia, a cell line originating from the distal tubular part of the kidney ofXenopus laevis, were cultured on permeable supports and mounted in an Ussing-type chamber. Cell thickness (T _c), short-circuit current (I _sc) and transepithelial conductance (G _t) were recorded while tissues were bilaterally incubated in NaCl solutions and the transepithelial potential was clamped to zero. Effects of inhibition and stimulation of transepithelial Na⁺ transport on cell volume and on its regulation during a hyposmotic challenge were investigated. Under control conditions a slow spontaneous decrease ofT _c described by a linear baseline was recorded. The reduction of the apical osmolality from 260 to 140 mosmol/kg did not alter cell volume significantly, demonstrating a negligible water permeability of the apical barrier. The inhibition of Na⁺ uptake by replacing apical Na⁺ byN-methyl-d-glucamine (NMDG⁺) did not affect cell volume under isotonic conditions. An increase ofT _c by 12.1% above the control baseline was recorded after blocking active transport with ouabain for 60 min. The activation of Na⁺ transport with insulin or oxytocin, which is known to activate the apical water permeability in other epithelia, did not alter cell volume significantly. The insensitivity of cell volume to alterations in apical Na⁺ uptake or Na⁺ pump rate confirms the close coupling between apical and basolateral transport processes. The blockage of basolateral K⁺ channels by 5 mM Ba²⁺ elicited a significant increase inT _c of 16.3% above control. Quinine, a potent blocker of volume-activated K⁺ channels, did not changeT _c significantly. Basolateral hypotonicity elicited a rapid rise inT _c followed by a regulatory volume decrease (RVD). An RVD was also recorded after blocking apical Na⁺ uptake as well as after stimulating apical Na⁺ uptake with oxytocin or insulin. Inhibition of active transport with ouabain as well as blocking K⁺ efflux at the basolateral side with Ba²⁺ or quinine abolished the RVD. The inhibition of the RVD by ouabain seems to be caused by a depletion of cellular K⁺, whereas the effects of Ba²⁺ and quinine are most likely due to the blockage of the basolateral K⁺ pathway.     

Oxytocin stimulates the apical K+ conductance in frog skin

We measured the effects of oxytocin (0.1 U/ml) on the current (I _sc) recorded through skins ofRana temporaria incubated with an isotonic K⁺ solution on the api al side while the transepithelial potential was clamped to zero. Under these conditions,I _sc is carried by inward K⁺ movements. Oxytocin markedly stimulated this inward K⁺ current. When the spontaneous fluctuations were analyzed we found that oxytocin increased the plateau (S _o ⁽¹⁾) of the spontaneous Lorentzian component without modifying the corner frequency (f _c ⁽¹⁾). Addition of Ba²⁺ to the mucosal solution blockedI _sc both in the presence and absence of oxytocin. Moreover, with mucosal Ba²⁺ a characteristic blocker-induced Lorentzian component appeared in the power spectrum. Analysis of this blocker-induced noise showed that oxytocin increased the number of active K⁺ channels in the apical membrane, while the changes in single channel current were in agreement with expected alterations of the electrochemical driving force.     

Ca2+- and swelling-induced activation of ion conductances in bronchial epithelial cells

The present study was performed to examine Ca²⁺-dependent and cell-swelling-induced ion conductances in a polarized bronchial epithelial cell line (16HBE14o-). Whole-cell currents were measured in fast and slow whole-cell patch-clamp experiments in cells grown either on filters or on coated plastic dishes. In addition the transepithelial voltage (V _te) and resistance (R _te) were measured in confluent monolayers. Resting cells had a membrane voltage (V _m) of –36±1.1 mV (n=137) which was mainly caused by K⁺ and Cl^– conductances and to a lesser extent by a Na⁺ conductance. V _te was apical-side-negative after stimulation. Equivalent short-circuit current (I _sc = V _te/R _te) was increased by the secretagogues histamine (0.1 mmol/l), bradykinin (0.1–10 mol/l) and ATP (0.1–100 mol/l). The histamine-induced I _sc was blocked by either basolateral diphenhydramine (0.1 mmol/l, n=4) or apical cimetidine (0.1 mmol/l, n=4). In fast and slow whole-cell recordings ATP and bradykinin primarily activated a transient K⁺ conductance and hyperpolarized V _m. This effect was mimicked by the Ca²⁺ ionophore ionomycin (1 mol/l, n=11). Inhibition of the bradykinin-induced I _sc by the blocker HOE140 (1 mol/l, n=3) suggested the presence of a BK₂ receptor. The potency sequence of different nucleotide agonists on the purinergic receptor was UTP ATP > ITP > GTP CTP [,-methylene] ATP 2-methylthio-ATP = 0 and was obtained in I _sc measurements and patch-clamp recordings. This suggests the presence of a P_2u receptor. Hypotonic cell swelling activated both Cl^– and K⁺ conductances. The Cl^– conductance was only slightly inhibited by 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (0.5 mmol/ l, n=3). These data indicate that 16HBE140- bronchial epithelial cells, which are known to express high levels of cystic fibrosis transmembrane conductance regulator protein, form a secretory epithelium. While hypotonic cell swelling activates both K⁺ and Cl^– channels, the Ca²⁺-induced Cl^– secretion is due mainly to activation of basolateral K⁺ channels.     

Chloride and potassium conductances of cultured human sweat ducts

The purpose of this study was to characterize the ion conductances, in particular those for Cl^– and K⁺, of human sweat duct cells grown in primary culture. Sweat duct cells from healthy individuals were grown to confluence on a dialysis membrane, which was then mounted in a mini-Ussing chamber and transepithelial and intracellular potentials were measured under open-circuit conditions. Under control conditions the epithelia developed mucosa-negative transepithelial potentials, V _te, of about –10mV. The apical membrane potential, V _a, was –25 mV to –30 mV (n=97) in most cells, but several cells had a higher potential of about –55 mV (n=29). Mucosal amiloride (10 mol/l) hyperpolarized V _a from –31±1 mV to a new sustained level of –46±2 mV (n=36). These changes were accompanied by increase in the fractional resistance of the apical membrane, fR _a, and decreases of V _te and the equivalent short-circuit current, I _sc. In amiloride-treated tissues an increase in mucosal K⁺ concentration (5 mmol/l to 25 mmol/l) depolarized V _a by 5±1 mV (n=8), while the same step on the serosal side depolarized V _a by 20±2 mV (n=8). A Cl^– channel blocker 3,5-dichloro-diphenylamine-2-carboxylate DCl-DPC; 10 mol/l) depolarized V _a by 5±1 mV (n=6), an effect that was lost after amiloride application. The blocker had no effect from the serosal side. Reduction of mucosal Cl^– (from 120 to 30 or 10 mmol/l) depolarized V _a by 9–11 mV (n=35), an effect that was often followed by a secondary hyperpolarization of 10–30 mV (n=27). Isoproterenol (5 mol/l) increased the V _a responses to low Cl^– such that the depolarizing response was increased from 10±1 mV to 19±2 mV (n=8); the hyperpolarizing response seemed to be reduced. With changes in Cl^– concentration on the serosal side, V _a remained relatively constant at –25 mV, while V _te decreased from –8 mV to–3 mV; hence, V _bl depolarized by about 5 mV. Taken together, our results show that the human sweat duct epithelium possesses Na⁺, K⁺ and Cl^– conductances on the luminal membrane and Cl^– and K⁺ conductances on the basolateral membrane. The Cl^– conductances on the luminal membrane is sensitive to DCl-DPC, and can be activated by isoproterenol. The small K⁺ conductance on the luminal membrane could account for some K⁺ secretion in sweat glands.     

Ion transport and electrophysiology of the early proximal colon of rabbit

The ion transport properties of the mammalian descending colon have been the subject of numerous investigations during the last decade. In contrast, relatively few studies have investigated proximal segments of this organ. In the present study, we assessed transepithelial transport of Na⁺, K⁺ and Cl^– in the isolated initial segment (P1) of rabbit colon in vitro using radioisotopic tracer fluxes and electrophysiological techniques. Like the rabbit descending colon, the proximal colon actively absorbs sodium and chloride, howeveer, its transport systems are markedly different. In vivo, this segment absorbs potassium, however in vitro active potassium secretion was observed. Unlike the descending colon, Na⁺ absorption is relatively insensitive to amiloride and only a slight inhibition was obtained even at 1 mM concentrations of this drug. Na⁺ and Cl^– absorption appeared to be coupled (directly or indicrectly) since the absorption of each ion was inhibited by the removal of the other. Serosal ouabain also inhibited Na⁺ and Cl^– absorption and net K⁺ secretion. Unlike the descending colon, the proximal P1 segment did not have a net absorptive K⁺ transport system that was detectable in the presence of ouabain. Electrically, the early proximal colon has a low transepithelial resistance compared to descending colon (R _T=133±7 cm²) but a larger short-circuit current (l _sc=178±12 A/cm²). The transepithelial potential averaged –21±1 mV, in excellent agreement with values measured in vivo. The apical and basolateral membrane potentials averaged –21±1 mV and –42±1 mV and intracellular potassium activity was 70±2 mM. The findings indicate active K⁺ uptake across the basolateral membrane and passive exit across the apical membrane. The basolateral membrane conductance may be a potassium conductance that is blockable by barium. It is likely that K⁺ transport normally occurs by both cellular and paracellular routes in this epithelium. Because of the numerous differences between this segment and the descending colon, we conclude that the P1 segment of proximal colon has a distinct function in colonic electrolyte transport     

Mechanism of NaCl secretion in the rectal gland of spiny dogfish (Squalus acanthias)

Rectal gland tubules (RGT) of spiny dogfish were dissected and perfused in vitro. Transepithelial PD (PD_te), resistance (R_te), the PD across the basolateral membrane (PD_bl) and intracellular chloride and potassium activities (a _Cl– ^cell ,a _K+ ^cell ) were measured. In a first series, 67 RGT segments were perfused with symmetric shark Ringers solution. The bath perfusate contained in addition db-cAMP 10^–4, forskolin 10^–6, and adenosine 10^–4 mol · l^–1. PD_te was –11±1 (n=67) mV lumen negative, R_te 27±2 (n=47) cm². PD_bl –75±0.4 (n=260) mV.a _K+ ^cell anda _Cl– ^cell were 109±22 (n=4) and 38±4 (n=36) mmol · l^–1 respectively. These data indicate that Cl^– secretion across the RGT must be an uphill transport process, whereas secretion of Na⁺ could be driven by the lumen negative PD_te. Intracellular K⁺ is 14 mV above equilibrium with respect to the basolateral membrane PD and Cl^– is 23 mV above equilibrium across the apical membrane. In series 2, the conductivity properties of the apical and basolateral membrane as well as that of the paracellular pathway were examined in concentration step experiments. Decrease of the basolateral K⁺ concentration led to a rapid hyperpolarization of PD_bt with a mean slope of 19 mV per decade of K⁺ concentration change. Addition of 0.5 mmol · l^–1 Ba²⁺ to the bath solution lead to a marked depolarization and abolished the response to K⁺ concentration steps. In the lumen a Cl^– concentration downward step led to a depolarization of the lumen membrane; resulting in a mean slope of 18 mV per decade of Cl^– concentration change. When dilution potentials were generated across the epithelium, the polarity indicated that the paracellular pathway is cation selective. In series 3 the equivalent short circuit current (I_sc=PD_te/R_te) was determined as a function of symmetrical changes in Na⁺ concentration, with Cl^– held at 276 mmol · l^–1, and as a function of symmetrical changes in Cl^– concentration, with Na⁺ held at 278 mmol · l^–1 I_sc was a saturable function of Na⁺ concentration (Hill coefficient 0.9±0.1,K _1/2 4.4 mmol · l^–1,n=7) and also a saturable function of Cl^– concentration (Hill coefficient 2.0±0.1,K _1/2 75 mmol · l^–1,n=11). These data are compatible with the assumption that the carrier responsible for NaCl uptake has a 1 Na⁺ per 2 Cl^– stoichiometry. In series 4, the effect of a K⁺ concentration downward step on PD_bl anda _Cl– ^cell transients was followed with high time resolution in the presence and absence of basolateral furosemide (5 · 10^–5 to 10^–4 mol · l^–1) in an attempt to examine whether K⁺ reduction on the bath side inhibits Na⁺Cl^– uptake by the carrier system as does e.g. furosemide. The data indicate that removal of K⁺ from the bath side exerts an effect comparable to that of furosemide, i.e. it inhibits the carrier. We conclude that NaCl secretion in the RGT cell comprises at the least the following components: In the basolateral membrane, the (Na⁺+K⁺)-ATPase, probably the Na⁺ 2 Cl^–K⁺ carrier, and a K⁺ conductance. In the apical membrane a Cl^– conductance; and a Na⁺ conductive paracellular pathway.Supported by Deutsche Forschungsgemeinschaft DFG-Gr 480/8-1. Parts of this study have been presented at the 3rd International Symposium on Ion Selective Electrodes, Burg Rabenstein 1983, 16th Annual Meeting American Society of Nephrology, Washington DC 1983, 49th Tagung der Deutschen Physiologischen Gesellschaft, Dortmund 1984. A summary of the present study was published in Bulletin Mount Desert Island Biological Laboratory (Vol. 83)     

Piretanide-dextran and piretanide-polyethylene glycol interact with high affinity with the Na+ 2 Cl− K+ cotransporter in the thick ascending limb of the loop of Henle

Piretanide blocks the Na⁺ 2Cl^– K⁺ cotransporter protein in the thick ascending limb (TAL) of the loop of Henle reversibly. When tested from the luminal side in isolated perfused cTAL segments it leads to a half maximal inhibition (IC₅₀) of the equivalent short circuit current (I_sc) at a concentration of 10^–6 mol/l. From the basolateral side it has no effect on I_sc up to 10^–4 mol/l. The present study was designed to search for high affinity blockers of the Na⁺ 2Cl^– K⁺ cotransporter with large molecular weight in an attempt to use these macromolecules for antibody-labelling or affinity separation of this transport-protein. Amino-ethyl-dextran or amino-ethyl-polyethylene glycol (M.W. 5kd) were coupled to isothiocyanato-piretanide (ISO-PIR) at room temperature in DMSO. The resulting compounds dextran-sulfonylurea-piretanide (PIR-DEX) and polyethylene glycol-sulfonylurea-piretanide (PIR-PEG) (M.W. 5.38kd) were purified and tested in isolated perfused cTAL segments. IC₅₀ values for ISO-PIR, PIR-DEX and PIR-PEG were estimated from dose response curves after their addition to the lumen or bath perfusate, respectively. ISO-PIR, PIR-DEX and PIR-PEG acted from the lumen side at 3·10^–6, 6·10^–6 and 2·10^–6 mol/l. The inhibitory effect was easily reversible. From the basolateral side no effect for any compound was seen at up to 10^–4 mol/l. In clearance experiments PIR-DEX was given to female Wistar rats as an i.v. bolus (25 mol/kg) and the diuretic urine was collected. After dialysis (exclusion limit 2.5kd) the dialysed urine and the dialysate were tested in isolated perfused cTAL segments. The dialysates had no effect on I_sc, but the dialysed urine inhibited I_sc by 35% from the luminal side. The present data show: High molecular derivatives of piretanide with dextran or polyethylene glycol moieties block the Na⁺ 2Cl^– K⁺ cotransporter in cTAL segments at roughly the same low concentration as piretanide itself. Our data exclude a metabolism of these piretanide compounds in the kidney. Since these macromolecular probes can probably not enter the cell their inhibitory effect indicates that the binding site for piretanide diuretics on the Na⁺ 2Cl^– K⁺ cotransporter is exposed on the surface of the luminal cell membrane.This study was supported by Deutsche Forschungsgemeinschaft Gr 480/9     

Inhibition of voltage-dependent Na+ and K+ currents by forskolin in nodes of Ranvier

The effect of forskolin on voltage-activated Na⁺ and K⁺ currents in nodes of Ranvier from the toad, Bufo marinus, has been examined using the vaseline-gap voltageclamp technique. Peak Na⁺ currents (I _Na) were reduced by 35% and the rate of decline of Na⁺ current during continuous depolarization was accelerated following treatment with 450 M forskolin. However, the voltage-dependence of steady-state inactivation as well as the rate of recovery from fast inactivation remained unchanged. Upon repetitive depolarization at 1–10 Hz, a further inhibition of I _Na (60%) was observed. This use-dependent or phasic inhibition recovers slowly at -80 mV ( 13 s) and had a voltage-dependence like that of activation of the Na conductance. Near maximal steady-state phasic inhibition occurred with depolarizing pulse durations of only 4 ms, consistent with a direct involvement of the open Na⁺ channel in the blocking process. Inhibition of the delayed K⁺ current (I _K) was characterized by a concentration-dependent reduction in steady-state current amplitude (IC₅₀ 80 M) and a concentration-independent acceleration of current inactivation. A similar inhibition of I _K was obtained with 1,9-dideoxyforskolin, a homolog which does not activate adenylate cyclase (AC). The results suggest that the inhibition of I _K and perhaps I _Na follows directly from drug binding and is not a consequence of AC activation.     

Kinetic properties of Na+/H+ exchange in cultured bovine pigmented ciliary epithelial cells

Uptake studies with²²Na were performed in cultured bovine pigmented ciliary epithelial cells, in order to characterize mechanisms of Na⁺ transport. A large part of Na⁺ uptake was sensitive to amiloride, quinidine and harmaline. Na⁺ uptake was stimulated by intracellular acidification (using the NH ₄ ⁺ prepulse technique), and was inhibited with increasing extracellular proton concentration. Decreasing extracellular pH from 7.5 to 7.0 increased the apparentK _M for Na⁺ from 38 to 86 mM without considerable changes inV _max. In the presence of 5 mM Na⁺ half maximal inhibition of amiloride sensitive Na⁺ uptake by extracellular protons was observed at a hydrogen concentration of 50 nM. In the presence of 50 mM Na⁺ the proton concentration necessary for 50% inhibition was 139 nM. Thus, the mode of inhibition of extracellular H⁺ seemed to be competitive with aK _i of 20–40 nM. 10 M amiloride increased the apparentK _M for Na⁺ from 33 mM to 107 mM, whileV _max remained nearly unchanged. IC₅₀ for amiloride was 6 M at 5 mM Na⁺ and 36 M in the presence of 150 mM Na⁺. Thus, amiloride behaves as a competitive inhibitor with aK _i of about 5 M. The affinities of Na⁺ to the transport site (K _M16 mM), to the inhibitory site for protons (K _M21 mM), and to the inhibitory site for amiloride (K _M26 mM) were in the same order of magnitude.In summary, we have presented evidence for the presence of a Na⁺/H⁺ exchanger in cultured bovine pigmented ciliary epithelial cells. The kinetic data suggest the presence of only one common extracellular binding site for Na⁺, H⁺ and amiloride.     

Characterisation of volume-activated ion transport across epithelial monolayers of human intestinal T84 cells

The effects of hypo-osmolarity upon transepithelial ion transport in human intestinal cell layers have been investigated. Exposure of the basal-lateral surfaces to hypo-osmotic media resulted in a transient stimulation of inward short-circuit current (I _sc). This transient stimulation of inward current by hypo-osmotic media was abolished by 100 mol/l 4,4-diisothiocyanostilbene 2,2-disulphonic acid (DIDS). After prestimulation of inward I _sc by vasoactive intestinal peptide (VIP) or by combinations of carbachol and prostaglandin E₁ hypoosmotic exposure of the basal-lateral surfaces resulted in a further transient stimulation of I _sc. The stimulation of I _sc in these conditions was largely insensitive to DIDS inhibition. Exposure of the basal-lateral surfaces to hypo-osmotic media resulted in a stimulation of loop-diuretic-insensitive ⁸⁶Rb efflux across the basal-lateral surfaces. In addition, hypo-osmotic exposure of T₈₄ cells is also associated with an increase in cytosolic Ca²⁺. It is concluded that the effects of hypo-osmotic exposure of T₈₄ cells on secretory I _sc are consistent with the activation of a DIDS-sensitive apical Cl^– conductance and a basal-lateral K⁺ conductance. With prior activation of inward I _sc by VIP via a cAMP-activated DIDS-insensitive apical Cl^– conductance, augmentation of the secretory current by hypo-osmotic exposure is likely to result primarily from increased basal-lateral K⁺ current and loop-diuretic-sensitive Cl^– uptake.     

Electrophysiological characterization of rabbit distal convoluted tubule cell

The distal convoluted tubule (DCT) from rabbit kidney were perfused in vitro to study the conductive properties of the cell membranes by using electrophysiological methods. When the lumen and the bath were perfused with a biearbonate free solution buffered with HEPES, the transepithelial voltage (V _T) averaged –2.8±0.6 mV (n=20), lumen negative. The basolateral membrane voltage (V _B) averaged –77.8±1.1 mV (n=33) obtained by intracellular impalement of microelectrodes. Cable analysis performed by injecting a current from perfusion pipette revealed that the transepithelial resistance was 21.8±1.7 ·cm² and the fractional resistance of the luminal membrane was 0.78±0.03 (n=8), indicating the existence of ionic conductances in the luminal membrane. Addition of amiloride (10^–5 mol/l) to the luminal perfusate or Na⁺ removal from the lumen abolished the lumen negativeV _T and hyperpolarized the apical membrane. An increase in luminal K⁺ concentration from 5 to 50 mmol/l reduced the apical membrane potential (V _A) by 37.5±2.6 mV (n=7), whereas a reduction of Cl^– in the luminal perfusate did not changeV _A significantly (0.5±0.5 mV,n=4). Addition of Ba²⁺ to the lumen reducedV _A by 42.6±1.0 mV (n=4). When the bathing fluid was perfused with 50 mmol/l K⁺ solution, the basolateral membrane voltage (V _B) fell from –76.8±1.5 to –31.0±1.3 mV (n=18), and addition of Ba²⁺ to the bath reducedV _B by 18.3±4.8 mV (n=7). Although a reduction of Cl^– in the bathing fluid from 143 to 5 mmol/l did not cause any significant fast initial depolarization (1.8±1.7 mV,n=8), a spike like depolarization (14.0±2.5 mV,n=4) was observed, upon Cl^– reduction in the presence of Ba²⁺ in the bath. From these results, we conclude that the apical membrane of DCT has both K⁺ and Na⁺ conductances and the basolateral membrane has a K⁺ conductance and a small Cl^– conductance.     

Electrophysiological study of transport systems in isolated perfused pancreatic ducts: properties of the basolateral membrane

In order to study the mechanism of pancreatic HCO ₃ ^– transport, a perfused preparation of isolated intra-and interlobular ducts (i.d. 20–40 m) of rat pancreas was developed. Responses of the epithelium to changes in the bath ionic concentration and to addition of transport inhibitors was monitored by electrophysiological techniques. In this report some properties of the basolateral membrane of pancreatic duct cells are described. The transepithelial potential difference (PD_te) in ducts bathed in HCO ₃ ^– -free and HCO ₃ ^– -containing solution was –0.8 and –2.6 mV, respectively. The equivalent short circuit current (I_sc) under similar conditions was 26 and 50 A·cm^–2. The specific transepithelial resistance (R_te) was 88 cm². In control solutions the PD across the basolateral membrane (PD_bl) was –63±1 mV (n=314). Ouabain (3 mmol/l) depolarized PD_bl by 4.8±1.1 mV (n=6) within less than 10 s. When the bath K⁺ concentration was increased from 5 to 20 mmol/l, PD_bl depolarized by 15.9±0.9 mV (n=50). The same K⁺ concentration step had no effect on PD_bl if the ducts were exposed to Ba²⁺, a K⁺ channel blocker. Application of Ba²⁺ (1 mmol/l) alone depolarized PD_bl by 26.4±1.4 mV (n=19), while another K⁺ channel blocker TEA⁺ (50 mmol/l) depolarized PD_bl only by 7.7±2.0 mV (n=9). Addition of amiloride (1 mmol/l) to the bath caused 3–4 mV depolarization of PD_bl. Furosemide (0.1 mmol/l) and SITS (0.1 mmol/l) had no effect on PD_bl. An increase in the bath HCO ₃ ^– concentration from 0 to 25 mmol/l produced fast and sustained depolarization of PD_bl by 8.5±1.0 mV (n=149). It was investigated whether the effect of HCO ₃ ^– was due to a Na⁺⁺-dependent transport mechanism on the basolateral membrane, where the ion complex transferred into the cell would be positively charged, or whether it was due to decreased K⁺ conductance caused by lowered intracellular pH. Experiments showed that the HCO ₃ ^– effect was present even when the bath Na⁺ concentration was reduced to a nominal value of 0 mmol/l. Similarly, the HCO ₃ ^– effect remained unchanged after Ba²⁺ (5 mmol/l) was added to the bath. The results indicate that on the basolateral membrane of duct cells there is a ouabain sensitive (Na⁺+K⁺)-ATPase, a Ba²⁺ sensitive K⁺ conductance and an amiloride sensitive Na⁺/H⁺ antiport. The HCO ₃ ^– effect on PD_bl is most likely due to rheogenic anion exit across the luminal membrane.     

Effect of high NaCl intake on Na+ and K+ transport in the rabbit distal convoluted tubule

Morphological studies have demonstrated that a chronic increase in distal Na⁺ delivery causes hypertrophy of the distal convoluted tubule (DCT). To examine whether high NaCl-intake also causes functional changes in the well defined DCT, we measured transmural voltage (V _T), lumen-to-bath Na⁺ flux (J _Na(LB)), and net K⁺ secretion (J _K(net)) in DCTs obtained from control rabbits and those on high NaCl-intake diets. The lumen negativeV _T was significantly greater in the high NaCl group than in the control group. The net K⁺ secretion (pmol mm^–1 min^–1) was greater in the high NaCl-intake group (54.1±13.0 vs 14.7±5.6). The K⁺ permeabïlities in both luminal and basolateral DCT membranes, as assessed by the K⁺-induced transepithelial voltage deflection inhibitable with Ba²⁺, were increased in the experimental group. The lumen-to-bath²²Na flux (pmol mm^–1 min^–1) was also greater in the experimental group (726±119 vs 396±65). TheV _T component inhibitable with amiloride was also elevated in the high NaCl-intake group. Furthermore, Na⁺–K⁺-ATPase activity of the DCT was higher in the experimental than in the control group. We conclude that high NaCl intake increases both Na⁺ reabsorption and K⁺ secretion by the DCT. This phenomenon is associated with an increased Na⁺–K⁺-ATPase activity along with increased Na⁺ and K⁺ permeabilities of the luminal membrane, and an increase in the K⁺ permeability of the basolateral membrane. Cellular mechanisms underlying these functional changes remain to be established.     

Sodium movement into and out of corneal endothelium

Rabbit corneal endothelial cells mounted in vitro were impaled simultaneously with Na⁺-selective and conventional KCl-filled microelectrodes. The membrane potential (V _m) was –30.4±0.8 mV (mean ±SEM, n = 55) and the intracellular [Na⁺]_i (calculated from the Na⁺-selective electrode potential, V_Na) was 13.7 ±1.9 mM (mean±SEM, n = 16). When ouabain was added to the perfusate the cell depolarised, causing both V _m and V_Na to increase with a very similar time course. Final V _m was –6.3±0.6 mV (mean ±SEM, n = 15), and the final [Na⁺]_i was 114±6.9 mM (mean ± SEM, n = 5). The parallel increase in V _m and rise in [Na⁺]_i suggest that a component of the ouabain-induced depolarisation of the cell (increase in V _m) is due to Na⁺ entry into the cell down its concentration gradient. The lateral and basal location of the Na⁺/K⁺-ATPase in bovine endothelial cells was confirmed (for the first time at the electron-microscopic level) using a monoclonal antibody specific for the 1 subunit of Na⁺/K⁺-ATPase. The absence of a net Na⁺ flux across these cells combined with the basolateral location of the ATPase suggest that Na⁺ exit from the cell, and its re-entry take place across the same membrane (i. e. the basolateral).